In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora but can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides diffiicile, independently uses or manipulates mucus to its benefit. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualization of an established genome-scale metabolic network reconstruction (GENRE). We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight a flexibility in metabolism that may influence pathogenesis.
Overall design: To assess the trasncriptional response of C. difficile to gastrointestinal mucus, we supplemented a C. difficile minimal medium (CDMM; from Cartman and Minton, Applied Environmental Microbiology, 2010) with 50 µg/mL mucus. C. difficile cultures were grown to exponential phase (OD600 ~0.5) before collection, RNA isolation, and RNA-sequencing.
Mucus was derived from primary human epithelial cells or porcine gastric mucus. Mucus was purified using 3K NMWL filters (Amicon), resuspended in PBS, and its total protein concentration quantified using Pierce BCA Assay to enable standardization. Only mucus from primary human epithelial cells was considered in the study above, as this mucus came from a more clinically relevant source, but reads from samples supplemented with commercial porcine gastric mucus (Type III, Sigma) are included here for public record.
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